Methanotrophy under extreme alkalinity in a serpentinizing system

Serpentinization produces hyperalkaline, H₂- and CH₄-rich fluids that support microbial life and serve as analogs for ocean worlds such as Enceladus. While methane production in these systems has been well studied, methane consumption-especially under high pH-remains poorly understood. Here, we present isotopic, geochemical, and genomic evidence for hyperalkaliphilic (pH > 11) methanotrophy in the Samail ophiolite of Oman. Using models that account for fluid mixing and gas exsolution, we identify δ¹³CH₄ enrichment that cannot be explained by abiotic processes alone. The enrichment of ¹³CH₄ co-occurs with methanotroph 16S rRNA gene sequences, particularly in fluids formed by mixing CH₄-rich, reduced fluids with oxidant-rich waters. Shotgun metagenome sequencing reveals a metagenome-assembled genome affiliated with Methylovulum, encoding a complete methane oxidation pathway, multiple carbon assimilation routes, and Na⁺/H⁺ antiporters-adaptations likely enabling growth above pH 11. Our findings highlight the viability of methanotrophy under extreme high pH conditions and provide a framework for interpreting δ¹³CH₄ signals in serpentinizing environments on Earth and beyond.